Agar Colony Research Articles

Antiadherent AgBDC Metal-Organic Framework Coating for Escherichia coli Biofilm Inhibition.

Surface microbial colonization and its potential biofilm formation are currently a major unsolved problem, causing almost 75% of human infectious diseases. Pathogenic biofilms are capable of surviving high antibiotic doses, resulting in inefficient treatments and, subsequently, raised infection prevalence rates. Antibacterial coatings have become a promising strategy against the biofilm formation in biomedical devices due to their biocidal activity without compromising the bulk material. Here, we propose for the first time a silver-based metal-organic framework (MOF; here denoted AgBDC) showing original antifouling properties able to suppress not only the initial bacterial adhesion, but also the potential surface contamination. Firstly, the AgBDC stability (colloidal, structural and chemical) was confirmed under bacteria culture conditions by using agar diffusion and colony counting assays, evidencing its biocide effect against the challenging E. coli, one of the main representative indicators of Gram-negative resistance bacteria. Then, this material was shaped as homogeneous spin-coated AgBDC thin film, investigating its antifouling and biocide features using a combination of complementary procedures such as colony counting, optical density or confocal scanning microscopy, which allowed to visualize for the first time the biofilm impact generated by MOFs via a specific fluorochrome, calcofluor.

Anticancer effects of gossypetin from Hibiscus sabdariffa in oral squamous cell carcinoma.

Gossypetin, isolated from Hibiscus sabdariffa L, has been shown to have various pharmacological effects including anti-inflammatory and antibacterial activity against various diseases. However, since the effect of gossypetin in oral cancer remains to be reported, we aimed to investigate the anticancer activity and mechanisms of gossypetin in oral squamous cell carcinoma (OSCC). The proliferation of OSCC cells was evaluated by cell viability and soft agar colony assays. The effects of gossypetin on the migration and invasion of OSCC cells was investigated by wound healing and transwell invasion assays, respectively. Apoptosis and cell cycle arrest were measured by flow cytometry. Moreover, the anticancer mechanism of gossypetin in OSCC cells was analyzed by western blotting. Gossypetin inhibited the proliferation, migration, and invasion of OSCC cells and induced apoptosis by upregulating the Bax/Bcl-2 ratio and cell cycle arrest at the G2/M phase. Furthermore, gossypetin regulated the activation of extracellular signal-regulated kinase and nuclear factor-kappa B. Results showed that gossypetin inhibits the proliferation, migration, and invasion of OSCC cells and triggers apoptosis and cell cycle arrest in OSCC. Therefore, gossypetin has the potential for use as a chemopreventive agent in oral cancer.

Inhibition of focal adhesion kinase/paxillin axis by caffeic acid phenethyl ester restrains aggressive behaviors of head and neck squamous cell carcinoma in vitro

ObjectiveCaffeic acid phenethyl ester (CAPE), one of the components of propolis that is produced by honeybees, reportedly suppresses multiple diseases, including bacterial infection, inflammation, and cancer. We aimed to investigate the inhibitory effects of CAPE on epithelial–mesenchymal transition (EMT) status and aggressive behaviors of human head and neck squamous cell carcinoma (HNSCC) in vitro and the underlying signaling pathway. DesignTo examine the cell growth and in vitro tumorigenic potential of HNSCC cells, cell viability and soft agar colony formation assays, respectively, were performed. Transwell migration and invasion assays were conducted to monitor HNSCC cells’ aggressive behaviors. Western blotting and immunocytochemistry analyses were done to investigate the signaling pathway responsible for relieving EMT progression and HNSCC cell aggressiveness. ResultsCAPE inhibited the in vitro tumorigenic potential of SNU-1041 cells stimulated by epidermal growth factor and suppressed the migratory and invasive capacities of SNU-1041 cells, irrespective of their cell proliferation state. CAPE was, at least partially, capable of inhibiting EMT progression by upregulating E-cadherin expression, which was accompanied by the reduction of phosphorylated focal adhesion kinase (FAK) and Paxillin. The inhibition of the FAK/Paxillin axis by PF-562271 was sufficient to alleviate the EMT progression through the induction of E-cadherin and aggressive behaviors of SNU-1041 cells. ConclusionsCAPE has a therapeutic potential as an anti-metastatic drug candidate for HNSCC therapy targeting the FAK/Paxillin axis.

Abstract A022: Limonene as a vehicle in topical delivery of carvedilol for skin cancer chemoprevention

Abstract Skin cancer is currently one of the most common cancers and is defined as an abnormal growth of the skin cells. The goal of this study is to develop a topical delivery system of carvedilol with limonene as the vehicle. Limonene is a major component found in the rinds of citrus fruits and other herbs. It is one of many natural compounds that have demonstrated inhibitory activity in different types of cancers such as breast, lung, and stomach cancer. Furthermore, limonene has shown to increase skin permeation of drugs including carvedilol. The soft agar colony formation assay was used to evaluate anchorage-independent growth of JB6 mouse epidermal cells. This assay tested concentrations of carvedilol (0.1, 1.0 and 10 μM); limonene (1.0, 10, 100 and 1000 μM); and a combination of carvedilol (1.0 μM) and limonene (1.0, 10 or 100 μM). Results demonstrated that carvedilol and limonene as single treatment inhibited colony formation. The combination of carvedilol and limonene showed higher colony inhibition than single treatment, although not statistically significant. Franz diffusion cell was used to test for permeation of the drug. The device has two chambers separated by a membrane, which in this case is rat skin. The drugs, in vehicles of 40% polyethylene glycol (PEG) 400 in PBS or 40% PEG400 in PBS plus 5% limonene were applied into the donor chamber while the samples are collected through the receptor chamber as the drug permeates through the skin. The samples were collected at 16 hours, 20 hours, and 24 hours. Results showed that limonene significantly increased permeation. Therefore, our data indicate that limonene was able to increase skin permeation and the chemopreventive activity of carvedilol. This new formulation will be further examined in animal models for its efficacy against UV-induced skin cancer. Citation Format: Janell Wang, Lawrence Chao, Steven Yeung, Ying Huang. Limonene as a vehicle in topical delivery of carvedilol for skin cancer chemoprevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A022.

Niclosamide Inhibits the Growth of Acute Lymphoblastic Leukemia Cells By Increasing Intracellular ROS and Activation of p53-PUMA/NOXA

Acute lymphoblastic leukemia (ALL) is a heterogeneous clonal malignancy originated from B- or T-cell lymphoid precursor cells. The prognosis is extremely poor in refractory or relapsed cases. Niclosamide is an FDA-approved oral anthelmintic drug with anti-cancer property. In the present study, we investigated the anti-leukemic effect of niclosamide in ALL cells in vitro and in vivo. ALL cell lines REH and NALM-6, and primary cells of patients with ALL were treated with niclosamide according to the concentration gradient, and then detected by trypan blue, MTS, and soft agar colony assays. The transcriptional and translational levels of apoptosis/anti-apoptosis-related genes were assessed by immunoblotting and quantitative real-time PCR. Flow cytometry was used to detect the effect of niclosamide on apoptosis of ALL cells, mitochondrial membrane potential, and intracellular specific reactive oxygen species (ROS) levels. For animal studies, we generated a mouse xenograft model with NOD prkdc-/-IL-2Rg-/- mice intravenously engrafted with NALM-6-luc cells, the NALM-6 cells infected with retrovirus expressing luciferase. Mice were treated with 25 mg/kg/day niclosamide or solvent twice a day intraperitoneally. The effect of niclosamide against leukemia cells in mice was dynamically evaluated by an in vivo imaging system. Flow cytometry was used to analyze the percentage of leukemia cells in spleens and bone marrows of sacrificed mice. The overall survival of mice was evaluated by Kaplan-Meier survival curve. In the cell viability assay, we found that niclosamide effectively inhibited REH, NALM-6 and primary ALL cells with IC50 values of 1.72, 3.58 and 1.36 μmol/L, respectively. Immunoblotting showed increased levels of pro-apoptotic proteins p53, PUMA, NOXA, cyto C and PARP, but decreased level of anti-apoptotic protein survivin in ALL cells treated with niclosamide in a dose- and time-dependent manner. Flow cytometry showed that niclosamide induced apoptosis and mitochondrial membrane potential in ALL cells. We also found that the level of ROS began to increase in cells treated by niclosamide for 2 hours, which reached the maximum at 8 hours. The treatment of ROS inhibitor N-acetylcysteine (NAC) attenuated niclosamide-induced apoptosis of REH and NALM-6 cells, which reduced by 18.12% and 8.02%, respectively. NAC also inhibited niclosamide-induced accumulation of intracellular p53/PUMA/NOXA proteins in these cells. In the animal study, in vivo imaging system showed significantly reduced tumor burdens in mice treated with niclosamide compared with controls, with bioluminescence signals of 15.53 and 4.04 on day 21, respectively. Mice treated with niclosamide survived longer than controls (32.5 days vs 26.5 days, p < 0.01), which had lower percentage of CD45+ CD19+ leukemia cells in their bone marrows and spleens, compared with the control group (61.10% vs 34.76% and 54.88% vs 26.40%, respectively). In the present study, we found that niclosamide potently inhibited the growth of ALL cells and induced apoptosis via increased levels of ROS and the activation of p53 signaling. Our findings suggest that niclosamide may be a promisingly potent agent for ALL therapy.

Inhibition of colorectal cancer tumorigenesis by ursolic acid and doxorubicin is mediated by targeting the Akt signaling pathway and activating the Hippo signaling pathway.

Colorectal cancer (CRC) is one of the deadliest malignant tumors worldwide and its prevalence is increasing in South Korea. The efficacy of combined treatment with natural product‑derived and chemotherapy agents including curcumin combined with 5‑fluorouracil, resveratrol combined with cisplatin and epigallocatechin‑3‑gallate (EGCG) combined with cisplatin in preventing cancer progression and killing cancer cells has emerged. The Akt and Hippo signaling pathways serve a key role in colorectal tumor growth; however, the exact role of the crosstalk between Akt and Hippo signaling pathways in CRC remains poorly elucidated. The combined effect of UA and DOX on the cell proliferation, apoptosis, migration and cell cycle of CRC cells were investigated by performing Cell proliferation assay, a soft agar colony formation assay, flow cytometry, wound healing assay and western blotting assay. Subsequently, the expression of AKT and Hippo signaling pathway‑associated proteins were also assessed by western blot assay. Moreover, a xenograft nude mouse model was constructed to verify the effects of UA and DOX on the tumorigenesis of HCT116 cell invivo. The present study reported that ursolic acid (UA) strongly enhanced the antitumor action of doxorubicin (DOX) via blocking the Akt/glycogen synthase kinase‑3β (Gsk3β) signaling pathway and activating tumor‑suppressive Hippo signaling (mammalian Ste20‑like kinase 1 and 2, salvador family WW domain containing protein 1 and MOB kinase activator 1), thereby downregulating downstream effector yes‑associated protein 1 (Yap) and connective tissue growth factor (CTGF) protein expression levels in CRC cells. Furthermore, The PI3K inhibitor LY294002 further suppressed Akt activity and enhance the function of Hippo pathway‑associated proteins in DOX + UA treated cells; this effect led to subsequent oncogenic Yap and CTGF inhibition following combined treatment, whereas Akt activator SC79 exerted an opposite effect in CTGF expression. Invivo, treatment with UA combined with DOX markedly suppressed the progression of CRC without any toxic effects on a xenograft mouse model by disrupting Akt signaling and activating the Hippo signaling pathway. These results demonstrated that UA and DOX treatment successfully induced Akt/Gsk3β inactivation via Hippo signaling pathway activation to promote Yap degradation, resulting in the inhibition of colorectal tumorigenesis. In conclusion, these findings suggested that combination therapy with UA and DOX may be more effective than DOX alone. UA may be a novel anticancer strategy and could be considered for investigation as a complementary chemotherapy agent in the future.

Skin Bacteriome and its Resistance to Antibiotics in Free Range Pigs

The cutaneous microbiome and also its resistance to antibiotics is exposed to change, depending on different habitat factors. This research investigates the composition of cutaneous microflora and its antibiotic resistance in pigs raised on free range farms (mix breed swine, from low input small farms). Swabs were collected from the skin surface and subjected to classical microbiological methods (simple broth and nutrient agar cultivation, colony isolation and biochemical API identification). The antibacterial resistance to gentamicin, streptomycin, oxitetracycline, tylosin, amoxacillin-clavulanic acid, marbofloxacin, tulatromycin, cefotaxime and doxycycline was estimated by Kirby Bauer method and multiple antibiotic resistance (MAR) index was calculated. Strains from Staphylococcus (sciuri and warnerii), Shigella spp., Kytococcus sedentarius, Salmonella spp. and Citrobacter freundii genera and species were identified in the collected samples. The most resistant was a S. warnerii strain, but the MAR index was high (0.33) in 50% of the strains. The most efficient antibiotic was cefotaxime and the least efficient was oxitetracycline. The results indicated the presence of antibiotic resistant ubiquitous and pathogenic strains in the investigated pigs which need caution, since they could express pathogenicity under appropriate conditions which low input farming system could provide.

Alpinumisoflavone Exhibits the Therapeutic Effect on Prostate Cancer Cells by Repressing AR and Co-Targeting FASN- and HMGCR-Mediated Lipid and Cholesterol Biosynthesis.

Prostate cancer (PCa) is the most common cancer in men, and this has been mainly noticed in Western and Asian countries. The aggregations of PCa and castration-resistant PCa (CRPC) progression are the crucial causes in the mortality of patients without the effective treatment. To seek new remedies for the lethal PCa diseases is currently an urgent need. In this study, we endeavored to investigate the therapeutic efficacy of alpinumisoflavone (AIF), a natural product, in PCa. LNCaP (androgen- sensitive) and C4-2 (CRPC) PCa cells were used. An MTT-based method, soft agar colony forming assay, biological progression approaches were applied to determine cell viability, migration, and invasion. A fatty acid quantification kit, a cholesterol detection kit and oil red O staining were conducted to analyze the intracellular levels of lipids and cholesterols. Apoptosis assays were also performed. AIF reduced cell viability, migration, and invasion in PCa cells. The expression of androgen receptor (AR), fatty acid synthase (FASN), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) was substantially inhibited by AIF treatment in PCa cells. Furthermore, by inhibiting FASN and HMGCR expression, AIF decreased the amounts of intracellular fatty acids, cholesterols, and lipid droplets in PCa cells. Significantly, through coordinated targeting FASN- and HMGCR-regulated biosynthesis and the AR axis, AIF activated the caspase-associated apoptosis in PCa cells. These results collectively demonstrated for the first time the potential of AIF as a novel and attractive remedy and provided an alternative opportunity to cure PCa malignancy.

Capnocytophaga sputigena Bacteremia in an Immunocompromised Child, When to Suspect? A Case Report and Review of the Literature

Background Within febrile neutropenic episodes, bacteremia is the most frequent cause. Capnocytophaga species bacteremia represents less than 3% of all etiologies, being an uncommon agent. Only a few pediatric cases have been reported in the literature. Case Presentation A 6-year-old male patient was diagnosed with acute lymphoblastic leukemia and a bone marrow relapse. He was admitted to our institution with an episode of febrile neutropenia with grade II mucositis. A nonhemolytic gram-negative rod was isolated from a blood culture, with gray colonies in blood agar at 48 hours of incubation. Capnocytophaga sputigena was identified by matrix-assisted laser desorption/ionization and time of flight. The patient was successfully treated with imipenem-cilastatin for 10 days. Conclusions Although this pathogen is uncommon, Capnocytophaga species bacteremia should be suspected as a causal agent in immunocompromised patients, particularly in cases of febrile neutropenia with oral cavity involvement.

Implications and Efficacy of Aromatase Inhibitors in Combination and Monotherapy for the Treatment of Lung Cancer.

Lung tumors express high levels of aromatase enzyme compared to surrounding normal tissue. Inhibition of aromatase has emerged as a recent therapeutic approach for the treatment of breast cancer. However, the role of aromatase inhibition in lung cancer treatment requires further investigation. The anti-proliferative effects of aromatase inhibitors were evaluated by MTT assay. Cell migration was assessed using a wound healing assay. The mechanism of cell death was determined using the annexin VFITC/ propidium iodide staining flow cytometry method. The soft agar colony formation assay evaluated cells' capability to form colonies. Exemestane and curcumin significantly inhibited the growth of lung cancer cell lines in a dose- and timedependent manner. The IC50 values after 48 hours of treatment with exemestane were 176, 180, and 120 μM in A549, H661, and H1299, respectively. Curcumin IC50 values after 48 hours were 80, 43, and 68 μM in A549, H661, and H1299, respectively. The combined treatment of exemestane or curcumin with cisplatin, raloxifene, and celecoxib resulted in a synergistic effect in the A549 lung cell line with a combination index of less than 1, suggesting synergism. Exemestane resulted in approximately 96% inhibition of wound closure at 100 μM, while curcumin resulted in approximately 63% inhibition of wound closure at 50 μM. Exemestane and curcumin inhibited the formation of cell colonies by reducing the number and size of formed colonies of A549, H661, and H1299 cell lines in a concentration dependent manner. Exemestane and curcumin had significantly induced apoptosis in A549 cells compared to control of untreated cells. Aromatase inhibition by exemestane or curcumin had significantly inhibited the growth of lung cancer cell lines, synergized with cisplatin, raloxifene, and celecoxib, suppressed lung cancer cell migratory potential, induced apoptosis, and reduced colony formation of lung cancer cells.

Synthesis of Environmental Bioplastic Polyhydroxyalkanoate (PHA) from Waste Glycerol, Palm Oil and Different Concentrations of Glucose by A New Strain Propionibacterium Sp.

Propionibacteria have mainly been found in dairy products and on the skin of humans and animals. This genus classified as non-producing PHA but in this study we show that Propionibacterium sp. can produce PHA. A member of the genus Propionibacterium was isolated from soil. The isolate was identified based on its gram positive pleomorphic rods. Cells occur short chains, singly or in pairs, in V or Y configurations. On mineral salt agar colonies are semi-opaque, convex, glittering and often pigmented in orange in color, biochemical tests and 16SrDNA also done. The 16SrDNA analysis confirmed 85% identity to Propionibacterium (accession number NR 074675.1). The presence of 3HB was identified, based on the analysis of NMR. 13C and 1H NMR analysis confirmed that Propionibacterium sp was able to produce PHA.polymer. These confirm the results of GC. The isolate was then grown on media with waste glycerol (WG), palm oil, different concentration of glucose and one stage and two stages of cultivation. This lead to the conclusion that Propionibacterium is able to grow utilizing waste glycerol, palm oil and different concentration of glucose as the sole carbon source under limited conditions. The PHA content 3 (wt%) and the composition (mol%) and (86% 3HB) when used glucose 2% at frist stage cultivation. while the PHA content 13 (wt %) when used palm oil and 5 (wt%) when utilized (WG), as sole carbon source.

High throughput-screening of native herbal compounds identifies taccaoside A as a cytotoxic compound that mediates RAS signaling in cancer stem cells

BackgroundCancer stem cells (CSCs) are characterized by their ability to self-renew, to differentiate into multiple cell types and also drive tumor formation, altogether making them important cellular targets for therapeutic intervention. However, existing CSC-targeting drugs do not significantly improve clinical outcomes. More recently, preclinical studies of natural product-derived compounds have demonstrated their potential usefulness as a therapeutic cancer treatment through their cytotoxic actions on CSCs. PurposeHere, we identify CSC-specific compounds derived from natural products and characterize their putative mechanisms of action in CSCs. MethodsGlioblastoma stem cells (GSCs) were labeled with EGFP via homologous recombination and utilized for a high-throughput screen of 8,344 fractions from 386 herbal medicines. The fractions that extinguished EGFP fluorescence signal were then further characterized by LC-MS/MS. Next, several putative cytotoxic compounds were evaluated for their cytotoxic effects on GSCs, cancer cell lines and immortalized cells using a variety of methods to study cell proliferation (EdU incorporation assay), cell death (cleaved-Caspase-3 immunostaining), DNA damage (comet assay), mitochondrial membrane changes (JC-1 immunostaining), and tumor formation in vitro (soft agar colony forming assay). We also performed surface plasmon resonance analysis, western blotting, and immunohistochemistry to characterize the putative mechanisms underlying the cytotoxic effects of putative compounds on GSCs. Finally, we carried out xenograft tumor growth assays to study the cytotoxic potential of several candidates in vivo. ResultsOur high throughput screen led to the identification of the furostanol saponin taccaoside A and its two homologs from the rhizomatous geophyte Tacca. subflabellata that were cytotoxic to GSCs. Interestingly, the cytotoxic effect of taccaoside A on cell lines was significantly less compared to its homologs, owing to stereochemical differences of a carbon–carbon double bond between C-20 and C-22. Molecular studies revealed that taccaoside A binds to RAS to inhibit downstream effector signaling. Correspondingly, blockade of the interaction between taccaoside A and RAS abolished the inhibitory effect of this compound on CSCs. Furthermore, taccaoside A treatment was effective in limiting tumor cell growth in vivo. ConclusionOur study yielded an effective approach to screen for CSC-specific agents. Through this approach, we identified taccaoside A from the rhizomatous geophyte Tacca. subflabellata are cytotoxic to CSCs through a molecular mechanism that involves RAS binding and suppression of its downstream signaling. Our findings indicate taccaoside A is a potential lead compound for anti-CSC drug discovery.

SMYD3 regulates gastric cancer progression and macrophage polarization through EZH2 methylation.

SET and MYND domain-containing protein 3 (SMYD3), a known histone methyltransferase, was reported to regulate cancer pathogenesis. However, its role in gastric development and progression remains unclear. EZH2 methylation had been associated with cancer metastasis, but the EZH2 methylation status in gastric cancer (GC) is unknown. Here, we report that EZH2 K421 methylation was responsible for gastric cancer cell soft agar colony formation, in vivo metastasis, and macrophage polarization. Mechanically, we identified SMYD3 as the methyltransferase of EZH2 at K421 residue which accelerates EZH2 Ubiquitin proteasome degradation. Cell harboring non-methylated EZH2 mutants promotes gastric cancer cell metastasis. Taken together, our results showed that SMYD3-EZH2 axis restricts gastric cancer metastasis via integrating epigenetic signaling.

1-Deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.

Monomerization of Homodimeric Trefoil Factor 3 (TFF3) by an Aminonitrile Compound Inhibits TFF3-Dependent Cancer Cell Survival.

Trefoil factor 3 (TFF3) is a secreted protein with an established oncogenic function and a highly significant association with clinical progression of various human malignancies. Herein, a novel small molecule that specifically targets TFF3 homodimeric functions was identified. Utilizing the concept of reversible covalent interaction, 2-amino-4-(4-(6-fluoro-5-methylpyridin-3-yl)phenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (AMPC) was identified as a molecule that interacted with TFF3. AMPC monomerized the cellular and secreted TFF3 homodimer at the cysteine (Cys)57-Cys57 residue with subsequent more rapid degradation of the generated TFF3 monomers. Hence, AMPC treatment also resulted in cellular depletion of TFF3 with consequent decreased cell viability in various human carcinoma-derived TFF3 expressing cell lines, including estrogen receptor positive (ER+) mammary carcinoma (MC). AMPC treatment of TFF3 expressing ER+ MC cells significantly suppressed total cell number in a dose-dependent manner. Consistently, exposure of TFF3 expressing ER+ MC cells to AMPC decreased soft agar colony formation, foci formation, and growth in suspension culture and inhibited growth of preformed colonies in 3D Matrigel. AMPC increased apoptosis in TFF3 expressing ER+ MC cells associated with decreased activity of EGFR, p38, STAT3, AKT, and ERK, decreased protein levels of CCND1, CCNE1, BCL2, and BCL-XL, and increased protein levels of TP53, CDKN1A, CASP7, and CASP9. siRNA-mediated depletion of TFF3 expression in ER+ MC cells efficiently abrogated AMPC-stimulated loss of cell viability and CASPASE 3/7 activities. Furthermore, in mice bearing ER+ MC cell-generated xenografts, AMPC treatment significantly impeded xenograft growth. Hence, AMPC exemplifies a novel mechanism by which small molecule drugs may inhibit a dimeric oncogenic protein and provides a strategy to impede TFF3-dependent cancer progression.

Growth Promotion Activity And Growth Pattern Of Pseudomonas Fluorescens On Different Solid Media

Study was conducted in vitro to study the growth promotion activity and growth pattern of Pseudomonas fluorescens on different solid media. Soil samples were collected randomly from rhizosphere of citrus plants for isolation of Pseudomonas. Twenty six isolates was isolated, out of twenty six isolates eight isolates showed abiliity of siderophore production, thirteen isolates showed positiveness for IAA production, nine isolates showed phosphate solubilization and seven isolates were positive for HCN production. For growth pattern study of P. fluorescens three different media i.e. King’s B, Pseudomonas agar and nutrient agar used. All the isolates showed fast growth on King’s B followed by Pseudomonas agar and nutrient agar. Mostly Pseudomonas fluorescens produced greenish yellow coloured colonies on King’s B and Pseudomonas agar and dull yellowish coloured colony and creamy white coloured colony on nutrient agar. Fluorescent pigmentation was observed on King’B and Pseudomonas agar medium.

The inhibitory effect and mechanism of Yi-qi-hua-yu-jie-du decoction on the drug resistance of gastric cancer stem cells based on ABC transporters

BackgroundThe drug resistance of tumor stem cells is an obstacle in gastric cancer (GC) treatment and the high expression of ABC transporters is a classic reason for drug resistance. This study aimed to construct a reliable GC drug-resistant stem cell model and explore the inhibitory effect and mechanism of Yi-qi-hua-yu-jie-du medicated serum (YQHY) on the drug resistance of GC stem cells based on ABC transporters.MethodsThe tumor stemness biomarker CD44 was primary identification from WGCNA. The magnetic-activated cell sorting (MACS) method was used to separate CD44( +)BGC823/5-Fu (BGC823/5–Fu-CSCs) cells and the stemness characteristics were verified from multiple dimensions. Then, the drug resistance index and expression of ABC transporter genes MDR1 and MRP1 were detected in CD44(−)/CD44(+) cells. The inhibition and apoptosis rates of the cells administrated with YQHY or/and 5-Fu were calculated to confirm that YQHY can suppress the drug resistance of BGC823/5-Fu-CSCs. Afterwards, the effects of YQHY on the expression of MDR1 and MRP1 and the activation of the PI3K/Akt/Nrf2 pathway were observed. Finally, under the administration of IGF-1 (the activator of PI3K/Akt pathway) and Nrf2 siRNA, the mechanism of YQHY on reversing the drug resistance of BGC823/5–Fu-CSCs through inhibiting the expression of MDR1 and MRP1 via PI3K/Akt/Nrf2 was verified.ResultsCD44 was a reliable GC stemness biomarker and can be applied to construct the drug-resistant GC stem cell model CD44(+)BGC823/5-Fu. The growth rate, cell proliferation index, soft agar colony formation, expression of stemness specific genes and tumorigenesis ability of CD44(+)BGC823/5-Fu cells were significantly higher than those of CD44(−)BGC823/5-Fu cells. BGC823/5–Fu-CSCs exhibited strong drug resistance to 5-Fu and high expression of ABC transporter genes MDR1 and MRP1 compared to CD44(-) cells. YQHY increased the inhibition and apoptosis rates to efficiently inhibit the drug resistance of BGC823/5–Fu-CSCs. Meanwhile, it suppressed the expression of MDR1 and MRP1 and restrained the activation of PI3K/Akt/Nrf2 signaling pathway. Finally, it was found that IGF-1 partially restored the activation of PI3K/Akt/Nrf2 pathway, alleviated the inhibition of MDR1 and MRP1, blocked the proliferation-inhibitory and apoptosis-promotion effects. YQHY and si-Nrf2 synergistically suppressed the MDR1/MRP1 expression and the drug resistance of BGC823/5–Fu-CSCs.ConclusionsCD44 was a reliable GC stemness biomarker, and the high expression of ABC transporter genes MDR1 and MRP1 was an important feature of drug-resistant stem cells. YQHY inhibited the MDR1 and MRP1 expression via PI3K/Akt/Nrf2 pathway, thus reversing the drug resistance of BGC823/5–Fu-CSCs.

Development of a fully automated platform for agar-based measurement of viable bacterial growth.

Dynamic in vitro antibacterial studies provide valuable insight on effective dosing strategies prior to translating to in vivo models. Frequent sampling is required to monitor the pharmacodynamics (PD) of these studies, leading to significant work when quantifying the bacterial load of the samples. Spreading a bacterial suspension on agar to allow colony counting is a proven process for measuring very low levels of growth, but commercial automation equipment to handle agar plating and colony counting at scale is not readily available. We describe a process to greatly decrease the hands-on time required for PD assays by utilizing general-purpose liquid handling robots to plate bacteria and a custom-made plate imager to automate colony counting. The platform developed handles the biological assay from beginning to end as well as sample tracking at each step of the process. The process relies heavily on custom automation scheduling software to enable dynamic process decisions and coordinate data flow throughout. Using the described platform, we can efficiently quantify >100 PD samples per day while maintaining the necessary dynamic range of the assay. Alleviating the main bottleneck in the dynamic antibacterial studies has allowed us to accelerate the rate of experiments to provide antibacterial dosing data within shorter timelines.

Effects of 1α,25-dihydroxyvitamin D3 and tacalcitol on cell signaling and anchorage-independent growth in T98G and U251 glioblastoma cells

The active hormonal form of vitamin D, 1α,25-dihydroxyvitamin D3, is reported to have 1000s of biological targets. The growth-suppressive properties of 1α,25-dihydroxyvitamin D3 and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1α,25-dihydroxyvitamin D3 and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indicated suppression of p70-S6 kinase levels by 1α,25-dihydroxyvitamin D3 and tacalcitol in T98G cells, whereas the levels of PLCγ, a target for phospholipid signaling, was slightly increased.Activation of STAT3, an important regulator of malignancy, was suppressed by 1α,25-dihydroxyvitamin D3 and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1α,24-dihydroxyvitamin D3), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1α,25-dihydroxyvitamin D3 and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumorigenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.

Cladosporium Leaf Spot Caused by Cladosporium variabile in Winter High Tunnel Production of Spinach (Spinacia oleracea) in Maine, United States.

In January 2021, leaf spots were observed on 1 of 22 proprietary spinach cultivars in a trial under high tunnels in Albion, ME. Approximately 1% of plants were symptomatic with <30% leaf area affected. Some spinach growers reported ≤100% incidence in high tunnels in the northeastern USA. Leaf spots on the cultivar in Albion were <5 mm in diameter and tan-colored with dematiaceous spores in short chains on long conidiophores at the center of older lesions. Lesions were surface-sterilized in 0.6% NaOCl for 90 s, rinsed in sterile water, dried, and placed on clarified V8 agar medium amended with chloramphenicol (100 µg/ml). Three Cladosporium isolates (Cds053, Cds055, and Cds057) produced olive-green colonies with dematiaceous conidia, 0- to 2-septate, catenulate, and 8.0-28.0 µm (16.6 ± 0.6 µm, mean ± standard error) x 4.0-12.0 µm (8.3 ± 0.2 µm) (n = 50/isolate). The partial internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α gene (tef1-α), and actin gene (act) were amplified and sequenced with primer sets UNUP18S42/UNLO28S576B (Bakkeren et al. 2000), EF1-728F/EF1-986R (Carbone and Kohn 1999), and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. The sequence of each locus was identical among the isolates. The tef1-α (GenBank Accession No. MZ905363) and act (MZ905362) sequences were identical to accessions of tef1-α (EF679480.1) and act (EF679556.1) of C. variabile (Cooke) G. A. de Vries, whereas the ITS rDNA sequence (MZ893461) was identical to that of many Cladosporium spp., including C. variabile. Each isolate was inoculated onto four replicate 42-day-old plants of each of the spinach cultivars Viroflay and Mandolin. The plants were enclosed in plastic bags for 24 h, atomized with a spore suspension (106 conidia/ml) prepared from potato dextrose agar colonies of each isolate, and returned to the bags for 24 h. Four plants of each cultivar were atomized with deionized water as a control treatment. Plants were arranged in a randomized complete block design. By 5 days, tan spots had developed on Cladosporium-treated plants of each cultivar. By 14 days, Mandolin had developed more severe lesions (20.0 ± 2.8%, mean ± standard error of leaf area with symptoms) than Viroflay (5.4 ± 0.4%). No control plants treated with water developed lesions. After 15 days, a leaf from a plant of each treatment was incubated in a moist chamber at 20˚C with a natural diurnal light cycle. Sporulation was observed at the center of lesions from Cladosporium-inoculated plants <24 h after incubation, and Cladosporium was re-isolated. The act locus amplified and sequenced from the re-isolates, as described above, had an identical sequence to that of the original isolates, fulfilling Koch's postulates. Empirically, Cladosporium leaf spot has established in winter high tunnel production of spinach in the northeastern USA (R. Katz, personal observation). To our knowledge, this is the first official documentation of the disease in Maine. The disease is favored by cool, humid conditions (Inglis et al. 1997). Management strategies under these conditions in Maine and greater New England include foliar applications of effective fungicides (du Toit et al. 2004), selection of partially resistant cultivars, cultural practices to reduce relative humidity and durations of leaf wetness, and planting pathogen-free or treated seed (du Toit and Hernandez-Perez 2005; Hernandez-Perez and du Toit 2006).